Proteins are conveniently produced in a variety of procaryotic and eucaryotic recombinant expression systems. For example, Eschericia coli-derived plasmid DNA vectors are widely used to express proteins both in vitro and in vivo. In vitro, such vectors are used for purposes ranging from e.g., preliminary evaluation of the nature of protein expression to large-scale manufacture of recombinant proteins. In vivo, DNA vectors are used, for example, for gene therapy and nucleic acid vaccination.
In general, effective vectors are those that express high levels of protein due to the use of efficient promoters and other control elements. Other factors that may contribute to efficient transfection of cells include: (1) uptake of plasmid by cells; (2) escape of plasmid from endocytic vesicles after endocytosis; (3) translocation of the plasmid from the cytoplasm into the nucleus; and (4) transcription of the plasmid in the nucleus.
Work from several laboratories suggests that a major barrier to efficient transfection is translocation of the plasmid into the nucleus, particularly in cells that do not undergo mitosis (e.g., myocytes). One parameter that may affect this step is the size of the plasmid, as the nuclear pore complex involved in uptake of macromolecules into the nucleus has a finite size. Hence, it is desirable to engineer small plasmids that retain the ability to express proteins at high levels. This has the potential to facilitate DNA delivery and allows the insertion of larger gene inserts than is feasible in larger plasmids. The latter point is particularly important for preparation of certain recombinant viral vectors that have a limited capacity to package plasmids, such as alphavirus and adeno-associated vectors.
One particularly effective system for the production of recombinant proteins employs vectors containing the human cytomegalovirus (hCMV) immediate-early (IE1) enhancer/promoter region which controls transcription of the immediate-early 72,000 molecular weight protein of hCMV. See, e.g., Chapman et al., Nuc. Acids Res. (1991) 19:3979-3986; and U.S. Pat. No. 5,688,688. The hCMV IE1 enhancer/promoter is one of the strongest enhancer/promoters known and is active in a broad range of cell types.
The hCMV IE1 enhancer/promoter region (FIG. 2) includes a tissue-specific modulator, multiple potential binding sites for several different transcription factors, and a complex enhancer. The transcribed region of the gene contains four exons and three introns. The largest of the introns, termed “Intron A,” is found within the 5′-untranslated region of the gene. See, e.g., Chapman et al., Nuc. Acids Res. (1991) 19:3979-3986 for the sequence and structure of this region in hCMV strain Towne, and Akrigg et al., Virus Res. (1985) 2:107-121, for a description of the corresponding region in hCMV strain AD169. The Intron A region of the hCMV IE1 enhancer/promoter has been shown to contain elements that enhance expression of heterologous proteins in mammalian cells. See, e.g., Chapman et al., Nuc. Acids Res. (1991) 19:3979-3986.
Introns are non-coding regions present in most pre-mRNA transcripts produced in the mammalian cell nucleus. Intron sequences can profoundly enhance gene expression when included in heterologous expression vectors. See, e.g., Buchman et al., Molec. Cell. Biol. (1988) 8:4395-4405; Chapman et al., Nuc. Acids Res. (1991) 19:3979-3986. Recent studies have demonstrated a connection between pre-mRNA splicing and export from the nucleus of mature mRNAs to the cytoplasm. Cullen, B. R., Proc. Natl. Acad. Sci. USA (2000) 97:4-6; and Luo et al., Proc. Natl. Acad. Sci. USA (1999) 96:14937-14942. Accordingly, increased levels of expression, such as those seen with the Intron A region of the hCMV IE1 enhancer/promoter, may be due to increased levels of translatable mRNAs in the cytoplasm.